Immunoglobulin Superfamily—Antibody Variable (V) Domains
Antibodies are the paradigm of specific high-affinity binding reagents and provide an antigen binding site by interaction of variable heavy (VH) and variable light (VL) immunoglobulin domains. The binding interface is formed by six surface polypeptide loops, termed complementarity determining regions (CDRs), three from each variable domain, which are highly variable and combined provide a sufficiently large surface area for interaction with antigen. Specific binding reagents can be formed by association of only the VH and VL domains into an Fv module. Bacterial expression is enhanced by joining the V-domains with a linker polypeptide into a single-chain scFv molecule. “Humanisation” of recombinant antibodies by grafting murine CDR loop structures onto a human Fv framework is disclosed by Winter et al EP-239400.
Methods to improve the expression and folding characteristics of single-chain Fv molecules were described by Nieba et al (1997). The properties of single V-domains, derived from natural mammalian antibodies, have been described by Gussow et al in WO/90/05144 and EP 0368684B1 and by Davis et al in WO/91/08482. Single camelid V-domains have been described by Hamers et al in WO/96/34103 and in WO/94/25591. A method for reducing the hydrophobicity of the surface of a human VH domain by replacing human amino acid sequences with camelid amino acid sequences was described by Davies and Riechmann (1994). Methods to exchange other regions of human VH sequences with camel sequences to further enhance protein stability, including the insertion of cysteine residues in CDR loops, were described by Davies and Riechmann (1996).
Several attempts to engineer high-affinity single domain binding reagents using either the VH or VL domains alone have been unsuccessful, due to lack of binding specificity and the inherent insolubility of single domains in the absence of the hydrophobic face where the VH and VL domains interact (Kortt et al, 1995).
T-cell Receptor Variable (V) Domains
The T-cell receptor has two V-domains that combine into a structure similar to the Fv module of an antibody that results from combination of the VH and VL domains. Novotny et al (1991) described how the two V-domains of the T-cell receptor (termed alpha and beta) can be fused and expressed as a single chain polypeptide and, further, how to alter surface residues to reduce the hydrophobicity directly analogous to an antibody scfv. Other publications describe the expression characteristics of single-chain T-cell receptors comprising two V-alpha and V-beta domains (Wulfing and Pluckthun, 1994; Ward, 1991).
Non-antibody Ligands—CTLA-4 and CD28 V-Like Domains
There are a class of non-antibody ligands which bind to specific binding partners which also comprise V-like domains. These V-like domains are distinguished from those of antibodies or T-cell receptors because they have no propensity to join together into Fv-type molecules. These non-antibody ligands provide an alternative framework for the development of novel binding moieties with high affinities for target molecules. Single domain V-like binding molecules derived from these non-antibody ligands which are soluble are therefore desirable. Examples of suitable non-antibody ligands are CTLA-4, CD28 and ICOS (Hutloff et al, 1999).
Cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) and the homologous cell-surface proteins CD28 and ICOS, are involved in T-cell regulation during the immune response. CTLA-4 is a 44 kDa homodimer expressed primarily and transiently on the surface of activated T-cells, where it interacts with CD80 and CD86 surface antigens on antigen presenting cells to effect regulation of the immune response (Waterhouse et al. 1996, van der Merwe et al. 1997). CD28 is a 44 kDa homodimer expressed predominantly on T-cells and, like CTLA-4, interacts with CD80 and CD86 surface antigens on antigen presenting cells to effect regulation of the immune response (Linsley et al. 1990). Current theory suggests that competition between CTLA-4 and CD28 for available ligands controls the level of immune response, for example, gene deletion of CTLA-4 in knock-out mice results in a massive over-proliferation of activated T-cells (Waterhouse et al. 1995).
Each CTLA-4 monomeric subunit consists of an N-terminal extracellular domain, transmembrane region and C-terminal intracellular domain. The extracellular domain comprises an N-terminal V-like domain (VLD; of approximately 14 kDa predicted molecular weight by homology to the immunoglobulin superfamily) and a stalk of about 10 residues connecting the VLD to the transmembrane region. The VLD comprises surface loops corresponding to CDR-1, CDR2 and CDR3 of an antibody V-domain (Metzler 1997). Recent structural and mutational studies on CTLA-4 suggest that binding to CD80 and CD86 occurs via the VLD surface formed from A′GFCC′ (SEQ ID NO: 139) V-like beta-strands and also from the highly conserved MYPPPY (SEQ ID NO: 1) sequence in the CDR3-like surface loop (Peach et al. 1994; Morton et al. 1996; Metzler et al. 1997). Dimerisation between CTLA-4 monomers occurs through a disulphide bond between cysteine residues (Cys120) in the two stalks, which results in tethering of the two extracellular domains, but without any apparent direct association between V-like domains (Metzler et al. 1997). Dimerisation appears to contribute exclusively to increased avidity for the ligands.
In vitro Expression of Soluble Forms of CTLA-4.
Neither the extracellular domains nor V-like domains (VLDs) of human CTLA-4 molecule have been successfully expressed as soluble monomers in bacterial cells, presumably due to aggregation of the expressed proteins (Linsley et al, 1995). Expression of the extracellular N-terminal domain (Met1 to Asp124, comprising Cys120) in E. coli results in production of a dimeric 28 kDa MW protein, in which two CTLA-4 V-like domains are joined by a disulphide linkage at Cys120. Truncation at Val114 removes these cysteines and was intended to enable expression of a 14 kDa VLD in soluble, monomeric form. However, the product aggregated and it was concluded that hydrophobic sites, which were normally masked by glycosylation, were now exposed and caused aggregation (Linsley et al, 1995).
There have been some reports of successful expression of monomeric, glycosylated CTLA-4 extracellular domains in eukaryotic expression systems (ie CHO cells and the yeast Pichia pastoris; Linsley et al. 1995; Metzler et al. 1997; Gerstmayer et al. 1997). Glycosylation in these eukaryotic expression systems is presumed to occur at the two N-linked glycosylation sites in the VLD (Asn76 and Asn108). However, high yields have only been described for expression of a gene encoding a CTLA-4 VLD fused to Ig-CH2/CH3 domains which produces a dimeric recombinant protein with 2 CTLA-4 VLDs attached to an Fc subunit (WO 95/01994 and AU 16458/95). AU 60590/96 describes mutated forms of CTLA-4 VLDs with single amino acid replacements of the first tyrosine (Y) in the MYPPPY (SEQ ID NO: 1) surface loop which retain and modifies the affinity for the natural CD80 and CD86 ligands. AU 60590/96 describes the preferred soluble form of CTLA-4 VLDs as a recombinant CTLA-4/Ig fusion protein expressed in eukaryotic cells and does not solve the aggregation problem in prokaryote expression systems. EP 0757099A2 describes the use of CTLA-4 mutant molecules, for example the effect of changes on ligand binding of mutations in the CDR3-like loop.